Over-expression of LSD1 promotes proliferation, migration and invasion in non-small cell lung cancer

Background

Lysine specific demethylase 1 (LSD1) has been identified and biochemically characterized in epigenetics, but the pathological roles of its dysfunction in lung cancer remain to be elucidated. The aim of this study was to evaluate the prognostic significance of LSD1 expression in patients with non-small cell lung cancer (NSCLC) and to define its exact role in lung cancer proliferation, migration and invasion.

Methods

The protein levels of LSD1 in surgically resected samples from NSCLC patients were detected by immunohistochemistry or Western blotting. The mRNA levels of LSD1 were detected by qRT-PCR. The correlation of LSD1 expression with clinical characteristics and prognosis was determined by statistical analysis. Cell proliferation rate was assessed by MTS assay and immunofluorescence. Cell migration and invasion were detected by scratch test, matrigel assay and transwell invasion assay.

Results

LSD1 expression was higher in lung cancer tissue more than in normal lung tissue. Our results showed that over-expression of LSD1 protein were associated with shorter overall survival of NSCLC patients. LSD1 was localized mainly to the cancer cell nucleus. Interruption of LSD1 using siRNA or a chemical inhibitor, pargyline, suppressed proliferation, migration and invasion of A549, H460 and 293T cells. Meanwhile, over-expression of LSD1 enhanced cell growth. Finally, LSD1 was shown to regulate epithelial-to-mesenchymal transition in lung cancer cells.

Conclusions

Over-expression of LSD1 was associated with poor prognosis in NSCLC, and promoted tumor cell proliferation, migration and invasion. These results suggest that LSD1 is a tumor-promoting factor with promising therapeutic potential for NSCLC.     

Phosphoproteomics identifies oncogenic Ras signaling targets and their involvement in lung adenocarcinomas

Background

Ras is frequently mutated in a variety of human cancers, including lung cancer, leading to constitutive activation of MAPK signaling. Despite decades of research focused on the Ras oncogene, Ras-targeted phosphorylation events and signaling pathways have not been described on a proteome-wide scale.

Methodology/Principal Findings

By functional phosphoproteomics, we studied the molecular mechanics of oncogenic Ras signaling using a pathway-based approach. We identified Ras-regulated phosphorylation events (n = 77) using label-free comparative proteomics analysis of immortalized human bronchial epithelial cells with and without the expression of oncogenic Ras. Many were newly identified as potential targets of the Ras signaling pathway. A majority (∼60%) of the Ras-targeted events consisted of a [pSer/Thr]-Pro motif, indicating the involvement of proline-directed kinases. By integrating the phosphorylated signatures into the Pathway Interaction Database, we further inferred Ras-regulated pathways, including MAPK signaling and other novel cascades, in governing diverse functions such as gene expression, apoptosis, cell growth, and RNA processing. Comparisons of Ras-regulated phosphorylation events, pathways, and related kinases in lung cancer-derived cells supported a role of oncogenic Ras signaling in lung adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells.

Conclusions/Significance

This study reveals phosphorylation events, signaling networks, and molecular functions that are regulated by oncogenic Ras. The results observed in this study may aid to extend our knowledge on Ras signaling in lung cancer.     

Fucoidan from Seaweed Fucus vesiculosus Inhibits Migration and Invasion of Human Lung Cancer Cell via PI3K-Akt-mTOR Pathways

Background

Recently there has been an increased interest in the pharmacologically active natural products associated with remedies of various kinds of diseases, including cancer. Fucoidan is a polysaccharide derived from brown seaweeds and has long been used as an ingredient in some dietary supplement products. Although fucoidan has been known to have anti-cancer activity, the anti-metastatic effects and its detailed mechanism of actions have been poorly understood. Therefore, the aims of this study were to demonstrate the anti-metastatic functions of fucoidan and its mechanism of action using A549, a highly metastatic human lung cancer cell line.

Methods and Principal Findings

Fucoidan inhibits the growth of A549 cells at the concentration of 400 µg/ml. Fucoidan treatment of non-toxic dose (0–200 µg/ml) exhibits a concentration-dependent inhibitory effect on the invasion and migration of the cancer cell via decreasing its MMP-2 activity. To know the mechanism of these inhibitory effects, Western blotting was performed. Fucoidan treatment down-regulates extracellular signal-related kinase 1 and 2 (ERK1/2) and phosphoinositide 3-kinase (PI3K)–Akt–mammalian target of rapamycin (PI3K-Akt-mTOR) pathways. Furthermore, fucoidan decreases the cytosolic and nuclear levels of Nuclear Factor-kappa B (p65).

Conclusions/Significance

The present study suggests that fucoidan exhibits anti-metastatic effect on A549 lung cancer cells via the down-regulation of ERK1/2 and Akt-mTOR as well as NF-kB signaling pathways. Hence, fucoidan can be considered as a potential therapeutic reagent against the metastasis of invasive human lung cancer cells.     

Opposing Functions of Akt Isoforms in Lung Tumor Initiation and Progression

Background

The phosphatidylinositol 3-kinase–regulated protein kinase, Akt, plays an important role in the initiation and progression of human cancer. Mammalian cells express three Akt isoforms (Akt1–3), which are encoded by distinct genes. Despite sharing a high degree of amino acid identity, phenotypes observed in knockout mice suggest that Akt isoforms are not functionally redundant. The relative contributions of the different Akt isoforms to oncogenesis, and the effect of their deficiencies on tumor development, are not well understood.

Methods

Here we demonstrate that Akt isoforms have non-overlapping and sometimes opposing functions in tumor initiation and progression using a viral oncogene-induced mouse model of lung cancer and Akt isoform-specific knockout mice.

Results

Akt1 ablation significantly delays initiation of lung tumor growth, whereas Akt2 deficiency dramatically accelerates tumorigenesis in this mouse model. Ablation of Akt3 had a small, not statistically significant, stimulatory effect on tumor induction and growth by the viral oncogene. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling and Ki67 immunostaining of lung tissue sections revealed that the delayed tumor induction in Akt1^−/− mice was due to the inhibitory effects of Akt1 ablation on cell growth and survival. Conversely, the accelerated growth rate of lung tumors in Akt2^−/− and Akt3^−/− mice was due to increased cell proliferation and reduced tumor cell apoptosis. Investigation of Akt signaling in tumors from Akt knockout mice revealed that the lack of Akt1 interrupted the propagation of signaling in tumors to the critical downstream targets, GSK-3α/β and mTOR.

Conclusions

These results demonstrate that the degree of functional redundancy between Akt isoforms in the context of lung tumor initiation is minimal. Given that this mouse model exhibits considerable similarities to human lung cancer, these findings have important implications for the design and use of Akt inhibitors for the treatment of lung cancer.     

RNA interferences targeting the Fanconi anemia/BRCA pathway upstream genes reverse cisplatin resistance in drug-resistant lung cancer cells

Background

Cisplatin is one of the most commonly used chemotherapy agent for lung cancer. The therapeutic efficacy of cisplatin is limited by the development of resistance.In this study, we test the effect of RNA interference (RNAi) targeting Fanconi anemia (FA)/BRCA pathway upstream genes on the sensitivity of cisplatin-sensitive (A549 and SK-MES-1) and -resistant (A549/DDP) lung cancer cells to cisplatin.

Result

Using small interfering RNA (siRNA), knockdown of FANCF, FANCL, or FANCD2 inhibited function of the FA/BRCA pathway in A549, A549/DDP and SK-MES-1 cells, and potentiated sensitivity of the three cells to cisplatin. The extent of proliferation inhibition induced by cisplatin after knockdown of FANCF and/or FANCL in A549/DDP cells was significantly greater than in A549 and SK-MES-1 cells, suggesting that depletion of FANCF and/or FANCL can reverse resistance of cisplatin-resistant lung cancer cells to cisplatin. Furthermore, knockdown of FANCL resulted in higher cisplatin sensitivity and dramatically elevated apoptosis rates compared with knockdown of FANCF in A549/DDP cells, indicating that FANCL play an important role in the repair of cisplatin-induced DNA damage.

Conclusion

Knockdown of FANCF, FANCL, or FANCD2 by RNAi could synergize the effect of cisplatin on suppressing cell proliferation in cisplatin-resistant lung cancer cells through inhibition of FA/BRCA pathway.     

Sequence-Dependent Antiproliferative Effects of Gefitinib and Docetaxel on Non-Small Cell Lung Cancer (NSCLC) Cells and the Possible Mechanism

Purpose

Recent clinical trials showed that the sequential combination of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and chemotherapy could prolong the PFS and/or OS of advanced non-small cell lung cancer (NSCLC) patients with EGFR mutation. The aim of present study was to assess the optimal combination sequence and to explore its possible mechanism.

Methods

PC-9 cells and A549 cells, the lung adenocarcinoma cells with mutant and wide-type EGFR respectively, were treated with docetaxel/gefitinib alone or in different combination schedules. The EGFR and K-ras gene status was determined by qPCR-HRM technique. Cell proliferation was detected by MTT assay. The expression and phosphorylation of EGFR, ERK, Akt and IGF-1R were detected by western blot. Cell cycle distribution was observed by flow cytometry.

Results

Only sequential administration of docetaxel followed by gefitinib (D→G) induced significant synergistic effect in both cell lines (Combination Index<0.9). The reverse sequence (G→D) resulted in an antagonistic interaction in both cell lines (CI>1.1), whereas the concurrent administration (D+G) showed additive (0.9<CI<1.1)-synergistic effect in PC-9 cells and antagonistic-additive effect in A549 cells. Mechanism studies showed that docetaxel-induced phosphorylation of EGFR and ERK was repressed by subsequently used gefitinib, but not by concurrent exposure of gefitinib. The gefitinib-repressed phosphorylation of EGFR and ERK was reversed neither by concurrent nor by subsequent administration of docetaxel. D+G reinforced their inhibition on the phosphorylation of IGF-1R in PC-9 cells.

Conclusions

The cytotoxic drugs followed by EGFR-TKIs may be the optimal combination for antiproliferative effects in EGFR-mutant NSCLC cells, and the phosphorylation of EGFR and ERK might contribute to this effect.     

Suppression of Lung Tumorigenesis by Leucine Zipper/EF Hand–Containing Transmembrane-1

Background

Leucine zipper/EF hand-containing transmembrane-1 (LETM1) encodes for the human homologue of yeast Mdm38p, which is a mitochondria-shaping protein of unclear function. However, a previous study demonstrated that LETM1 served as an anchor protein for complex formation between mitochondria and ribosome, and regulated mitochondrial biogenesis.

Methodology/Principal Findings

Therefore, we examine the possibility that LETM1 may function to regulate mitochondria and lung tumor growth. In this study, we addressed this question by studying in the effect of adenovirus-mediated LETM1 in the lung cancer cell and lung cancer model mice. To investigate the effects of adenovirus-LETM1 in vitro, we infected with adenovirus-LETM1 in A549 cells. Additionally, in vivo effects of LETM1 were evaluated on K-ras ^LA1 mice, human non-small cell lung cancer model mice, by delivering the LETM1 via aerosol through nose-only inhalation system. The effects of LETM1 on lung cancer growth and AMPK related signals were evaluated. Adenovirus-mediated overexpression of LETM1 could induce destruction of mitochondria of lung cancer cells through depleting ATP and AMPK activation. Furthermore, adenoviral-LETM1 also altered Akt signaling and inhibited the cell cycle while facilitating apoptosis. Theses results demonstrated that adenovirus-LETM1 suppressed lung cancer cell growth in vitro and in vivo.

Conclusions/Significance

Adenovirus-mediated LETM1 may provide a useful target for designing lung tumor prevention and treatment.     

Non-small cell lung carcinoma cell motility, rac activation and metastatic dissemination are mediated by protein kinase C epsilon

Background

Protein kinase C (PKC) ε, a key signaling transducer implicated in mitogenesis, survival, and cancer progression, is overexpressed in human primary non-small cell lung cancer (NSCLC). The role of PKCε in lung cancer metastasis has not yet been established.

Principal Findings

Here we show that RNAi-mediated knockdown of PKCε in H358, H1299, H322, and A549 NSCLC impairs activation of the small GTPase Rac1 in response to phorbol 12-myristate 13-acetate (PMA), serum, or epidermal growth factor (EGF). PKCε depletion markedly impaired the ability of NSCLC cells to form membrane ruffles and migrate. Similar results were observed by pharmacological inhibition of PKCε with εV1-2, a specific PKCε inhibitor. PKCε was also required for invasiveness of NSCLC cells and modulated the secretion of extracellular matrix proteases and protease inhibitors. Finally, we found that PKCε-depleted NSCLC cells fail to disseminate to lungs in a mouse model of metastasis.

Conclusions

Our results implicate PKCε as a key mediator of Rac signaling and motility of lung cancer cells, highlighting its potential as a therapeutic target.     

SHP-2 promotes the maturation of oligodendrocyte precursor cells through Akt and ERK1/2 signaling in vitro

Background

Oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes (OLs), which are responsible for myelination. Myelin is essential for saltatory nerve conduction in the vertebrate nervous system. However, the molecular mechanisms of maturation and myelination by oligodendrocytes remain elusive.

Methods and Findings

In the present study, we showed that maturation of oligodendrocytes was attenuated by sodium orthovanadate (a comprehensive inhibitor of tyrosine phosphatases) and PTPi IV (a specific inhibitor of SHP-2). It is also found that SHP-2 was persistently expressed during maturation process of OPCs. Down-regulation of endogenous SHP-2 led to impairment of oligodendrocytes maturation and this effect was triiodo-L-thyronine (T3) dependent. Furthermore, over-expression of SHP-2 was shown to promote maturation of oligodendrocytes. Finally, it has been identified that SHP-2 was involved in activation of Akt and extracellular-regulated kinases 1 and 2 (ERK1/2) induced by T3 in oligodendrocytes.

Conclusions

SHP-2 promotes oligodendrocytes maturation via Akt and ERK1/2 signaling in vitro.     

Mechanism of cigarette smoke condensate-induced acute inflammatory response in human bronchial epithelial cells

Background

To demonstrate the involvement of tobacco smoking in the pathophysiology of lung disease, the responses of pulmonary epithelial cells to cigarette smoke condensate (CSC) — the particulate fraction of tobacco smoke — were examined.

Methods

The human alveolar epithelial cell line A549 and normal human bronchial epithelial cells (NHBEs) were exposed to 0.4 μg/ml CSC, a concentration that resulted in >90% cell survival and <5% apoptosis. Changes in gene expression and signaling responses were determined by RT-PCR, western blotting and immunocytofluorescence.

Results

NHBEs exposed to CSC showed increased expression of the inflammatory mediators sICAM-1, IL-1β, IL-8 and GM-CSF, as determined by RT-PCR. CSC-induced IL-1β expression was reduced by PD98059, a blocker of mitogen-actived protein kinase (MAPK) kinase (MEK), and by PDTC, a NFκB inhibitor. Analysis of intracellular signaling pathways, using antibodies specific for phosphorylated MAPKs (extracellular signal-regulated kinase [ERK]-1/2), demonstrated an increased level of phosphorylated ERK1/2 with increasing CSC concentration. Nuclear localization of phosphorylated ERK1/2 was seen within 30 min of CSC exposure and was inhibited by PD98059. Increased phosphorylation and nuclear translocation of IκB was also seen after CSC exposure. A549 cells transfected with a luciferase reporter plasmid containing a NFκB-inducible promoter sequence and exposed to CSC (0.4 μg/ml) or TNF-α (50 ng/ml) had an increased reporter activity of approximately 2-fold for CSC and 3.5-fold for TNF-α relative to untreated controls.

Conclusion

The acute phase response of NHBEs to cigarette smoke involves activation of both MAPK and NFκB.     

Spry1 and Spry4 Differentially Regulate Human Aortic Smooth Muscle Cell Phenotype via Akt/FoxO/Myocardin Signaling

Background

Changes in the vascular smooth muscle cell (VSMC) contractile phenotype occur in pathological states such as restenosis and atherosclerosis. Multiple cytokines, signaling through receptor tyrosine kinases (RTK) and PI3K/Akt and MAPK/ERK pathways, regulate these phenotypic transitions. The Spry proteins are feedback modulators of RTK signaling, but their specific roles in VSMC have not been established.

Methodology/Principal Findings

Here, we report for the first time that Spry1, but not Spry4, is required for maintaining the differentiated state of human VSMC in vitro. While Spry1 is a known MAPK/ERK inhibitor in many cell types, we found that Spry1 has little effect on MAPK/ERK signaling but increases and maintains Akt activation in VSMC. Sustained Akt signaling is required for VSMC marker expression in vitro, while ERK signaling negatively modulates Akt activation and VSMC marker gene expression. Spry4, which antagonizes both MAPK/ERK and Akt signaling, suppresses VSMC differentiation marker gene expression. We show using siRNA knockdown and ChIP assays that FoxO3a, a downstream target of PI3K/Akt signaling, represses myocardin promoter activity, and that Spry1 increases, while Spry4 decreases myocardin mRNA levels.

Conclusions

Together, these data indicate that Spry1 and Spry4 have opposing roles in VSMC phenotypic modulation, and Spry1 maintains the VSMC differentiation phenotype in vitro in part through an Akt/FoxO/myocardin pathway.     

Mini Profile of Potential Anticancer Properties of Propofol

Background

Propofol (2, 6-diisopropylphenol) is an intravenous sedative-hypnotic agent administered to induce and maintain anesthesia. It has been recently revealed that propofol has anticancer properties including direct and indirect suppression of the viability and proliferation of cancer cells by promoting apoptosis in some cancer cell lines.

Methodology/Principal Findings

This study aimed to establish a profile to quantitatively and functionally evaluate the anticancer properties of propofol in three cancer cell lines: non-small cell lung carcinoma cell line A549, human colon carcinoma cell line LoVo, and human breast cancer cell line SK-BR-3. We demonstrated that the expression level of caspase-3, an apoptosis biomarker, significantly increased in a dose-dependent manner after 24-h stimulation with 100 µM propofol in A549 cells, and slightly increased in LoVo cells. However, there was no change in caspase-3 expression in SK-BR-3 cells. High caspase-3 expression in A549 cells may be modulated by the ERK1/2 pathway because phosphorylated ERK1/2 dramatically reduced after propofol treatment. BAX, a major protein that promotes apoptosis in the regulation phase, was highly expressed in A549 cells after treatment with 25 µM propofol. Apoptosis induced by propofol may be associated with cancer cells carrying Kras mutations.

Conclusions/Significance

Our results suggest that the anti-cancer effects of propofol, which are consistent with those of previous studies, are likely associated with the Kras mutation status. Only Kras mutation in Codon 12 instead of other Kras status has been demonstrated to play an important role in sensitizing the propofol-induced apoptosis in cancer cell lines from our study. These findings may enable us a detailed investigation of propofol/Kras-mediated cancer cell apoptosis in the future.     

AD-1, a novel ginsenoside derivative,shows anti-lung cancer activity via activation of p38 MAPK pathway and generation of reactive oxygen species

Background

Ginseng is a traditional Chinese herb that has been used for thousands of years. In the present study, effects and mechanisms of AD-1 were evaluated for its development as a novel anti-lung cancer drug.

Methods

The cytotoxic activity was evaluated by MTT assay. Flow cytometry was employed to detect cell cycle, apoptosis and ROS. Western blot and immunohistochemistry were used to analyze signaling pathways. Lung cancer xenograft models were established by subcutaneous implantation of A549 or H292 cells into nude mice.

Results

AD-1 concentration-dependently reduces lung cancer cell viability without affecting normal human lung epithelial cell viability. In A549 and H292 lung cancer cells, AD-1 induces G₀/G₁ cell cycle arrest, apoptosis and ROS production. The apoptosis can be attenuated by a ROS scavenger — N-acetylcysteine (NAC). In addition, AD-1 up-regulates the expression of p38 and ERK phosphorylation. Addition of a p38 inhibitor SB203580, suppresses the AD-1-induced decrease in cell viability. Furthermore, genetic silencing of p38 attenuates the expression of p38 and decreases the AD-1-induced apoptosis. Treatment with NAC reduces AD-1-induced p38 phosphorylation, which indicates that ROS generation is involved in the AD-1-induced p38 activation. In mice, oral administration of AD-1 (10–40 mg/kg) dose-dependently inhibited the growth of xenograft tumors without affecting body weight and decreases the expression of VEGF, MMP-9 and CD34 in tumor tissue. TUNEL staining confirms that the tumors from AD-1 treated mice exhibit a markedly higher apoptotic index.

Conclusions and general significance

These data support development of AD-1 as a potential agent for lung cancer therapy.     

Houttuynia cordata Thunb extract modulates G0/G1 arrest and Fas/CD95-mediated death receptor apoptotic cell death in human lung cancer A549 cells

Background

Houttuynia cordata Thunb (HCT) is commonly used in Taiwan and other Asian countries as an anti-inflammatory, antibacterial and antiviral herbal medicine. In this study, we investigated the anti-human lung cancer activity and growth inhibition mechanisms of HCT in human lung cancer A549 cells.

Results

In order to investigate effects of HCT on A549 cells, MTT assay was used to evaluate cell viability. Flow cytometry was employed for cell cycle analysis, DAPI staining, and the Comet assay was used for DNA fragmentation and DNA condensation. Western blot analysis was used to analyze cell cycle and apoptotic related protein levels. HCT induced morphological changes including cell shrinkage and rounding. HCT increased the G₀/G₁ and Sub-G₁ cell (apoptosis) populations and HCT increased DNA fragmentation and DNA condensation as revealed by DAPI staining and the Comet assay. HCT induced activation of caspase-8 and caspase-3. Fas/CD95 protein levels were increased in HCT-treated A549 cells. The G₀/G₁ phase and apoptotic related protein levels of cyclin D1, cyclin A, CDK 4 and CDK 2 were decreased, and p27, caspase-8 and caspase-3 were increased in A549 cells after HCT treatment.

Conclusions

The results demonstrated that HCT-induced G₀/G₁ phase arrest and Fas/CD95-dependent apoptotic cell death in A549 cells     

Effects of karanjin on cell cycle arrest and apoptosis in human A549, HepG2 and HL-60 cancer cells

Background

We have investigated the potential anticancer effects of karanjin, a principal furanoflavonol constituent of the Chinese medicine Fordia cauliflora, using cytotoxic assay, cell cycle arrest, and induction of apoptosis in three human cancer cell lines (A549, HepG2 and HL-60 cells).

Results

MTT cytotoxic assay showed that karanjin could inhibit the proliferation and viability of all three cancer cells. The induction of cell cycle arrest was observed via a PI (propidium iodide)/RNase Staining Buffer detection kit and analyzed by flow cytometry: karanjin could dose-dependently induce cell cycle arrest at G2/M phase in the three cell lines. Cell apoptosis was assessed by Annexin V-FITC/PI staining: all three cancer cells treated with karanjin exhibited significantly increased apoptotic rates, especially in the percentage of late apoptosis cells.

Conclusion

Karanjin can induce cancer cell death through cell cycle arrest and enhance apoptosis. This compound may be effective clinically for cancer pharmacotherapy.     

Valproic Acid Induces Cutaneous Wound Healing In Vivo and Enhances Keratinocyte Motility

Background

Cutaneous wound healing is a complex process involving several signaling pathways such as the Wnt and extracellular signal-regulated kinase (ERK) signaling pathways. Valproic acid (VPA) is a commonly used antiepileptic drug that acts on these signaling pathways; however, the effect of VPA on cutaneous wound healing is unknown.

Methods and Findings

We created full-thickness wounds on the backs of C3H mice and then applied VPA. After 7 d, we observed marked healing and reduced wound size in VPA-treated mice. In the neo-epidermis of the wounds, β-catenin and markers for keratinocyte terminal differentiation were increased after VPA treatment. In addition, α-smooth muscle actin (α-SMA), collagen I and collagen III in the wounds were significantly increased. VPA induced proliferation and suppressed apoptosis of cells in the wounds, as determined by Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining analyses, respectively. In vitro, VPA enhanced the motility of HaCaT keratinocytes by activating Wnt/β-catenin, ERK and phosphatidylinositol 3-kinase (PI3-kinase)/Akt signaling pathways.

Conclusions

VPA enhances cutaneous wound healing in a murine model and induces migration of HaCaT keratinocytes.     

Involvement of STIM1 and Orai1 in EGF-mediated cell growth in retinal pigment epithelial cells

Background

In non-excitable cells, one major route for calcium entry is through store-operated calcium (SOC) channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca²⁺ store. STIM1 and Orai1 are major regulators of SOC channels. In this study, we explored the functions of STIM1 and Orai1 in epidermal growth factor (EGF)-induced cell proliferation and migration in retinal pigment epithelial cells (ARPE-19 cell line).

Results

EGF triggers cell proliferation and migration in ARPE-19 cells. Cell proliferation and migration involve STIM1 and Orai1, as well as phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2, and Akt. Pharmacological inhibitors of SOC channels and siRNA of Orai1 and STIM1 suppress cell proliferation and migration. Pre-treatment of mitogen-activated protein kinase kinase (MEK) inhibitors and a phosphatidylinositol 3 kinases (PI3K) inhibitor attenuated cell proliferation and migration. However, inhibition of the SOC channels failed to prevent EGF-mediated ERK 1/2 and Akt phosphorylation.

Conclusions

Our results showed that STIM1, Orai1, ERK 1/2, and Akt are key determinants of EGF-mediated cell growth in ARPE-19 cells. EGF is a potent growth molecule that has been linked to the development of PVR, and therefore, STIM1, Orai1, as well as the MEK/ERK 1/2 and PI3K/Akt pathways, might be potential therapeutic targets for drugs aimed at treating such disorders.     

GRP78 knockdown enhances apoptosis via the down-regulation of oxidative stress and Akt pathway after epirubicin treatment in colon cancer DLD-1 cells

Introduction

The 78-kDa glucose-regulated protein (GRP78) is induced in the cancer microenvironment and can be considered as a novel predictor of responsiveness to chemotherapy in many cancers. In this study, we found that intracellular reactive oxygen species (ROS) and nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation were higher in GRP78 knockdown DLD-1 colon cancer cells compared with scrambled control cells.

Methodology/Principal Findings

Treatment with epirubicin in GRP78 knockdown DLD-1 cells enhanced apoptosis and was associated with decreased production of intracellular ROS. In addition, apoptosis was increased by the antioxidants propyl gallate (PG) and dithiothreitol (DTT) in epirubicin-treated scrambled control cells. Epirubicin-treated GRP78 knockdown cells resulted in more inactivated Akt pathway members, such as phosphorylated Akt and GSK-3β, as well as downstream targets of β-catenin expression. Knockdown of Nrf2 with small interfering RNA (siRNA) increased apoptosis in epirubicin-treated GRP78 knockdown cells, which suggested that Nrf2 may be a primary defense mechanism in GRP78 knockdown cells. We also demonstrated that epirubicin-treated GRP78 knockdown cells could decrease survival pathway signaling through the redox activation of protein phosphatase 2A (PP2A), which is a serine/threonine phosphatase that negatively regulates the Akt pathway.

Conclusions

Our results indicate that epirubicin decreased the intracellular ROS in GRP78 knockdown cells, which decreased survival signaling through both the Akt pathway and the activation of PP2A. Together, these mechanisms contributed to the enhanced level of epirubicin-induced apoptosis that was observed in the GRP78 knockdown cells.